Biochemical and immunological testing of cellular and particulate matter typically involves a series of discrete reactions to attain the desired end product. Between successive steps of the reaction series, the cellular or particulate matter typically needs to be washed thoroughly or transferred to a new reaction vessel, or both, prior to exposure to the reactants involved in the subsequent reaction step. Such precautions need to be followed in order to prevent the subsequent step or steps from being contaminated by unwanted fluid-phase components of earlier reactions. When reactions free from contamination are attained, the end product is either a fluid-phase or solid-phase entity which can be legitimately and reliably detected and quantified by such means as visual inspection, fluorescence, spectrophotometry, and radiography.
The attainment of legitimate and reliable end products critically depends on how well the solid-phase components can be isolated from fluid-phase components during washing and transfer procedures. Over the years, various methodologies have been developed to facilitate the separation of solids from fluids. Older methodologies include centrifugation, gravity filtration, vacuum-assisted filtration, gravity separation, adsorption, and a variety of differential separation techniques based on molecular weights, conformation, etc. In the case of centrifugation, separation and adsorption techniques, the fluid-phase needs to be decanted or otherwise drawn off from the isolated solid phase before transfer or further washing steps can be accomplished. These techniques are all time consuming and subject to contamination whenever removal or transfer of components occurs.
Filtration has multiple advantages over the other techniques listed above. First, filtration is generally less time consuming, especially when filtration is assisted by applying a vacuum across the base of the filter. Second, the need to decant or draw off the fluid phase is avoided (as is this potential source of contamination) by the choice of filtering material. When properly chosen, the filter material forms a mesh with a large surface area to bind the solid-phase biological coreactant while allowing the fluid phase to pass through unimpeded. Third, the solid material can be multiply washed while being bound by the solid-phase support, thereby both facilitating the speed and ease of the wash steps as well as minimizing the problem of contamination. Fourth, concentration of the coreactant bound to the support material both increases the efficiency of transfer of this material to subsequent reaction vessels and enhances detection of the final solid-phase reaction product.
Recent advances in biochemical micromethods have focused on the advantages offered by filtration. Prior to incorporation of filtration techniques, biochemical reactions were carried out in test tubes or, when simultaneous testing of multiple small samples was desired, in commerically available manifolds consisting of an array of small tubes held together in a grid pattern. The lack of filtration capabilities in these earlier methodologies meant that these techniques were constrained by the limitations imposed by centrifugation, adsorption, and separation technologies, as described above. These limitations have been overcome within the last five years by incorporation of new filtration techniques into existing biochemical micromethodologies.
The earliest of these was described by Cole and Van Voorhis (U.S. Pat. No. 4,407,943; Oct. 4, 1983). In this device, antigens are immobilized on a membrane composed of interconnecting networks of pores to provide a large surface area for reactions to occur. This membrane was attached across the base of the reaction well, such that reaction fluids placed within the reaction well then flowed, due to hydrostatic pressure, through the tortuous course of the membrane, and then dripped off the underside of the membrane into a waste fluid collection chamber. Rate of flow through the membrane, and hence the time allowed for fluid components to interact with the bound antigen, was regulated by the diameter of the membrane pores and the volume of reaction fluid (hence the amount of hydrostatic pressure attained as a driving force) placed in the reaction well.
Although the invention of Cole and Van Voorhis represents a definite step forward in micromethodology, more recent approaches have recognized that increased sensitivity can be achieved if the reaction fluids are retained within the reaction well until drawn out of the well by vacuum filtration.
To date, three major approaches have been taken to merge vacuum filtration capabilities with biochemical micromethods. Although differing in design (see below for details), these three approaches all share one primary concept; that is, replacing the non-permeable bottom of the reaction vessel with a permeable material which overlies a low-resistance port through which fluid can pass upon application of vacuum.
By this single design change, two major advances were attained over prior methodologies. First, upon addition of the reagents, the biochemical or immunological reaction commenced and continued within the vessel until such time as the fluid components were evacuated from the vessel by vacuum applied across the permeable base of the reaction vessel. This meant that the same vessel was used for the reaction as well as for the separation of the solid phase from the fluid phase. Second, transfer of the reaction product to a new vessel for each reaction step was obviated, due to the efficiency in washing the vessel and reaction product afforded by this technique. In sum, then, this basic design change enhanced the speed, efficiency and specificity of biochemical and immunologic reactions.
Although this basic design change was common to all three currently available variations of vacuum-based biochemical micromethodologies, the three differed significantly in their approaches, and each had its own constellation of strengths and weaknesses. The first vacuum-based biochemical micromethod was introduced by Cleveland (U.S. Pat. No. 4,427,415; Jan. 24, 1984). In the commerically available version of his device, Cleveland modified a standard 96-well (8.times.12 matrix; volume of each flat-bottomed well=approx 350 .mu.l) microtiter tray by piercing the center of each well bottom to form a small (approximately 22 gauge) drain hole. The size of this drain hole was such that the surface tension of the fluids within the well prevented flow through the hole until vacuum was applied across the base of the microtiter tray. Positioned within each well, directly above the drain hole and covering the entire well bottom, was a circular disk of Whatman 934/AH glass microfiber filter material. The filter material served to trap the solid-phase reaction product when the fluid phase was evacuated from the well interior through the filter under vacuum.
However, multiple problems were inherent in this Cleveland technique. First, the drain hole was small. While this restriction was needed in order to attain the necessary surface tension to retain fluid within the well, this restriction in turn produced a very low bubble point. That is, the fluid being evacuated under vacuum through the drain hole bubbled and sprayed as it exited, especially when vacuum pressure was elevated or when the fluid had any inherent tendency to foam, as is true for most washing or blocking buffers (i.e., protein-based buffers such as albumens, gelatins, or milk). This low bubble point had the marked disadvantage of contaminating the reactions occurring in neighboring wells, due to spread of reagents between wells.
Second, the Cleveland drain hole was small and was located only under the center of the filter. Since effective washing of the trapped solid-phase component and effective removal of unreacted substrate are both critical to the specificity of the final reaction product attained, the device gave rise to problems. It resulted in uneven washing, since the vacuum was highest directly over the drain hole and was minimal at the lateral edges of the circular filter. This, in turn, led to a disparity in the effectiveness of the wash steps in the center, as against lateral, regions of the filter, as well as producing a tendency for reaction fluids to become trapped between the lateral aspects of the filter and the non-permeable well base (lateral to the drain hole).
Third, the die-cut Whatman glass microfiber filters used by Cleveland were compressed, due to the manufacturing technique employed. This compression created regions which did not allow fluids to pass, thereby resulting in uneven deposition of the solid-phase reaction products and regions of ineffectual washing.
These problems were overcome, in part, by structures such as those of Cleveland (U.S. Pat. No. 4,427,415; Jan. 24, 1984) and Fernwood and Burd (U.S. Pat. No. 4,493,815; Jan. 15, 1985). In each of these devices, a modified microtiter tray was used, such that the base of each of the ninety-six wells was removed, leaving the cylindrical wells completely open on both ends. The open-ended wells were then placed upon a single large sheet of filter material, which, in turn, rested across the bases of all wells. The filter material, in turn, rested upon a plate containing ninety-six drain holes which were aligned with the centers of the ninety-six wells above the filter material. This "plate-filter-plate sandwich" was then tightly clamped together, so that the lowest-resistance pathway for fluid placed in the wells was through the filter material and out through the drain holes in the base plate. By extending the length of the drain holes, cross-contamination due to the low bubble point was minimized. However, waste fluids which remained within the elongated exit port could contact the base of the filter material and contaminate the reaction occurring within the overlying well. These devices did not address the problem of uneven washing, which arose due to the uneven vacuum pressure applied across the lateral extent of the filter. Additionally, the required use of sheets of filter material restricted the choice of filters to materials such as nitrocellulose and bioaffinity membranes which have unidirectional pores, totally negating the use of glass microfiber or paper filter materials which draw fluids laterally between wells with remarkable tenacity. This capillary action of the microfibers and paper fibers cannot be overcome by any of the above-mentioned designs.
The last, and most recent, of the three major advances prior to the present invention is exemplified by Kiovsky and Hendrick (U.S. Pat. No. 4,526,690; July 2, 1985). Their manifold was again designed to overcome inadequacies inherent in the previous designs, yet their manifold was plagued by its own unique problems. The Kiovsky and Hendrick plate employed a ninety-six well microtiter plate where, as in the plate described in the previous paragraph, the entire bases were removed from all wells. Heat sealed to all of the bases of the ninety-six wells was a set of three sheets. The innermost sheet (that is, the one in direct contact with the wells) was a nitrocellulose material and the outer two sheets (that is, those further from the wells) were colandered Tyvek. The non-wettable, hydrophobic characteristics of Tyvek allowed fluid to be retained within the wells in the absence of applied vacuum, and the colandered processing of the Tyvek allowed fluid to be drawn through this material under vacuum. The nitrocellulose material provided a surface for collecting solid-phase reaction products and was thin enough to allow heat sealing to occur between the microtiter plate material and Tyvek.
The major advantage of this Kiovsky and Hendrick modification was that even vacuum pressure was applied across the entire filter surface, instead of maximally over a small drain hole as in previous methods. The purpose was to provide a more uniform wash of solid-phase materials and removal of fluid-phase reactants. A second advantage of this modification was that filter material was not die cut; so there were no compressed regions resulting in uneven concentration of solid-phase products. A third advantage was that, though a bubble point was definitely attainable during normal usage of this device, it was higher than in the first device described, since the surface area over which vacuum was applied was much greater.
Despite these advantages, this Kiovsky and Hendrick manifold suffered from several flaws. First, the user was restricted to using fragile, paper-thin filter material like nitrocellulose, since thicker filter materials (such as paper or glass microfibers) prevented the manifold-to-Tyvek heat sealing from occurring. This presented a problem since nitrocellulose is inappropriate for many filtering needs due to the small (0.5-5.0 micron) pore sizes which clog during many applications. Second, the filter material was a single sheet bound to all ninety-six wells, which meant that the filter, and the solid-phase reaction product trapped upon it, could not be easily removed upon completion of the reaction procedure for further testing of the reaction product or storage of the test results. Although the filter could be cut away from each well upon completion of the reaction for further testing or storage, the nitrocellulose material is extremely fragile and does not lend itself to this procedure, but rather cracks and tears unevenly. Further, removal of single filters disrupted the integrity of the Tyvek base, which in turn destroyed the vacuum seal required for filtering. Fourth, cross-contamination between wells was a serious problem with this manifold design. Not only was the bubble point reached under normal usage (resulting in spraying and bubbling of fluid-phase components as they exited the wells), but also the use of a continuous sheet of Tyvek across the entire matrix of 96 wells provided a surface which the bubbles and spray could cling to, thereby increasing the probability of cross-contamination between wells. This cross-contamination occurred despite the fact that the heat-seal design of this plate should have theoretically decreased the chance of cross-contamination, since the heat-seal which ringed the base of each well was hydrophobic and tended to keep the fluid being evacuated from each well inside the hydrophobic ring. However, if fluid did cross this ring (as occurred when the plate was jostled during the reaction procedure or when the contents of any of the ninety-six wells bubbled during the vacuuming steps), the hydrophobic nature was destroyed and there was no resistance to fluid movement across the Tyvek sheet.
Published International patent application WO No. 82/03690 of Bjorkman shows a hydrophobic membrane locked into--but not sealed to--a reaction vessel. The vessel has an inturned lower end, above which is a frustoconical chamber portion leading to an inverted frustoconical portion of the vessel which widens out to a tapered, nearly cylindrical, wall. These specially shaped vessels are separate from a rack having a series of recesses, although the publication states that the rack's recesses may serve as reaction vessels; if so, they provide the needed porous bottom (the hydrophobic membrane), presumably again by trapping the membrane in an enlarged recess above an inturned lower end of each recess for no sealing is shown or discussed. On top of the porous membrane a "depth filter", which may be hydrophilic, is applied to the upper surface of the porous bottom, and covers the pores thereof. According to the last claim therein, his hydrophilic filter is "pressed against the upper surface of the vessel bottom". The porous membranes actually used are said to have been "made of teflon and hydrophobized bottoms of hydrophilic polymeric material."
All the recesses in the Bjorkman racks lead down into narrow channels; the recesses and the reaction vessels are not wide open at their lower ends. Moreover, it would be nearly impossible to manufacture such racks by injection molding or any other inexpensive process. The Bjorkman device is necessarily an expensive device, impractical for use in great quantity.